Search

Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) show significant synergism with cellulase in cellulose degradation. In recent years, there have been many reports on AA9 LPMOs; however, the identification of efficient and thermostable AA9 LPMOs with broad potential for industrial applications remains necessary. In this study, a new AA9 LPMO from Talaromyces cellulolyticus, named TcAA9A, was identified. An analysis of the oxidation products of phosphoric acid-swollen cellulose categorized TcAA9A as a type 3 AA9 LPMO, and TcAA9A exhibited a better synergistic effect with cellulase from Trichoderma reesei than what is seen with TaAA9A, a well-studied AA9 LPMO from Thermoascus aurantiacus. Two AA9 LPMOs were successfully expressed in T. reesei, and the transformants were named TrTcAA9A and TrTaAA9A. The activities and thermostabilities of the AA9 LPMOs in TrTcAA9A were higher than those of the AA9 LPMOs in TrTaAA9A or the parent. The enzyme solution of TrTcAA9A was more efficient than that of the parent or TrTaAA9A for the degradation of Avicel and delignified corncob residue. Thus, TcAA9A showed a better performance than TaAA9A in T. reesei cellulase cocktails. This study may offer an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation. [ABSTRACT FROM AUTHOR]

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium. [ABSTRACT FROM AUTHOR]

Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆ vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆ vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression. [ABSTRACT FROM AUTHOR]

Trichoderma reesei is the most important industrial cellulase-producing filamentous fungus. Although its molecular physiology has been investigated, the signal transduction pathways are not fully understood. In particular, the role of casein kinase II (CKII) is not yet clear. In this work, we carried out functional investigations on a catalytic subunit of CKII, CKIIα2. Comparison of the phenotypic features of T. reesei parent and Δck2α2 strains showed significant changes following ck2α2 disruption. T. reesei Δck2α2 form significantly smaller mycelial pellets in glucose-containing liquid minimum media, have shorter and fewer branch hyphae, produce smaller amounts of chitinases, produce more spores, show more robust growth on glucose-containing agar plates, and consume glucose at a significantly higher rate. Suggestions can be made that CKIIα2 governs chitinase expression, and the disruption of ck2α2 results in lower levels of chitinase production, leading to a weaker cell wall disruption capability, further resulting in weaker hyphal branching, which eventually leads to smaller mycelial pellets in liquid media. Further conclusions can be made that CKIIα2 is involved in repression of sporulation and glucose metabolism, which is consistent with the proposal that CKIIα2 represses global metabolism. These observations make the deletion of ck2α2 a potentially beneficial genetic disruption for T. reesei during industrial applications, as smaller mycelial pellets, more spores and more robust glucose metabolism are all desired traits for industrial fermentation. This work reports novel unique functions of a CKII catalytic subunit and is also the first genetic and physiological investigation on CKII in T. reesei. [ABSTRACT FROM AUTHOR]

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli β-glucuronidase ( gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50–100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100°C for 30 min. [ABSTRACT FROM AUTHOR]

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6′-phytase, which owns high specific activity. To achieve a high expression level of 6′-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor α-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene ( phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml−1. The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50°C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system. [ABSTRACT FROM AUTHOR]